Sodium (Na) channels are fundamental signaling molecules in excitable cells, and are principal molecular targets for the local anesthetic (LA) agents. As Na channels change their conformational state, or "gate" in response to membrane depolarization, the intensity of the drug-receptor interaction is increased ~ 100-fold,1 a phenomenon termed "use dependence."2 While use dependence underlies the broad therapeutic efficacy of LA agents, under pathophysiologic conditions causing cellular stress, these state-dependent binding interactions may also provoke life-threatening side effects, such as cardiac arrhythmias or seizures.3,4 Studies supported by this program have shown that slow inactivation, a gated conformational state associated with sustained or frequent cellular depolarization, plays a critical role in facilitating use-dependent LA action. However, translating this understanding into strategies to avoid toxic side effects requires an improved understanding of the three-way mechanistic linkage between cellular stress, Na channel gating, and LA action. Intracellular free Ca2+ rises when neuronal or cardiac cells are subjected to patholphysiologic stress, such as ischemia or traumatic injury. We have identified two mechanisms whereby intracellular Ca2+ is capable of modulating voltage-gated Na channel (Nayl) inactivation gating function, and both involve regions within the C-terminus. These include Ca2+ binding to an EF-hand domain6 and Ca2+-regulated binding of calmodulin (CaM) to an "IQ" motif.7 At the same time, our preliminary data show that raising intracellular free Ca2+ enhances use-dependent LA action by increasing slow inactivation. Further, we find that mutation of either the EF-hand or the IQ motif alters this Ca2+-regulated LA action. Related studies have shown the ayl C-terminal IQ motif interacts directly with at least two other Na channel cytoplasmic domains with potential roles in inactivation: an N-terminal CaM-binding region8 and the III-FV interdomain linker.9 Using a combination of methods informing structure and function, including NMR spectroscopy, site-directed mutagenesis, and patch-clamp electrophysiology of wild-type and mutated Na channels, we will examine the mechanistic interactions between Ca2+ signaling and LA action. In particular, we will test the hypothesis that intracellular Ca2+, CaM, and LA agents have shared binding interactions involving the Na channel C-terminus that are mechanistically coupled during slow inactivation gating and use-dependent LA action.